The long term objective of this award is to understand how human telomeres protect chromosome ends. We are examining the structure and function of human and mouse telomeres in molecular detail in hopes of providing mechanistic insights that could enhance opportunities for diagnosis and treatment of human aging and cancer. In the next funding period, our efforts will be focused on the consequences of telomere dysfunction, in particular the DNA repair reactions that act on dysfunctional telomeres. Initial studies had identified the fusion of telomeres as the main detrimental outcome of DNA repair at dysfunctional telomeres. Telomere fusion is a prominent outcome of telomere attrition and inhibition of TRF2, a key protective factor at telomeres. We have obtained genetic evidence that telomere fusions are mediated by non-homologous end-joining (NHEJ). We propose that telomeres are protected from NHEJ through formation of the t-loop, a structure that can be formed by interaction of TRF2 with telomeric DNA in vitro. Our recent data indicates that in addition to NHEJ, three other repair reactions may threaten chromosome ends: homologous recombination (HR) of t-loops resulting in telomere deletion, HR between telomeres resulting in sister telomere exchange, and recombination of telomeres with interstitial telomere related sequences. Our aim is to define of all four repair reactions and to determine how telomeres prevent these events. Our experiments will involve manipulating the function of TRF2 and its interacting partners using the genetic, molecular genetic, and cell biological tools we have developed during the past funding period. Our specific aims are: AIM 1. To define the NHEJ pathway at telomeres AIM 2. To define the t-loop HR pathway and test its relevance in transformed cells AIM 3. To define the pathway responsible for sister telomere exchanges AIM 4. To test whether telomeres can recombine with interstitial telomeric DNA. Since telomere dysfunction is thought to be the main source of genome instability in human cancer and may contribute to aspects of aging, the detailed understanding of the repair pathways at human dysfunctional telomeres is expected to be highly significant with regard to human disease.